We have developed and validated a method for the high throughput inhibition screening of the major human cytochromes P450 (CYP) using an invitro substrate cocktail and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis. A mixture of probe substrates comprising phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), amitriptyline (CYP2C19), chlorzoxazone (CYP2E1), bufuralol (CYP2D6) and midazolam (CYP3A4) was incubated with pooled human liver microsomes and measured by LC/MS/MS in a single incubation mixture. The assay was specific and reproducible with measured enzymatic parameters (Km and Vmax) for each substrate showing values similar to those reported in the literature. The IC50 values of each inhibitor in the cocktail were determined and agreed well with values previously reported in the literature. This assay was used to evaluate the drug-drug interaction potential of currently used anticancer drugs. Potent and specific inhibition of CYP2D6 enzyme was detected with 50μM vinblastine, whilst little or moderate inhibition of the metabolism of the specific probe substrates was found with other anticancer compounds tested. 17- Allylamino-17-demethoxygeldanamycin (17-AAG), a novel HSP90 inhibitor, showed significant inhibition of CYP2C9, CYP2C19 and CYP2D6 at 50μM. However, the inhibition constant (Ki) for 17-AAG was >10μM, suggesting that these interactions are unlikely to have any clinical relevance. The validated method described here offers an efficient, robust way to determine the CYP inhibition profile of large number of compounds and is now used to support our drug discovery process.